Defining the cellular metabolic responses to brain activity using fluorescent biosensors

项目摘要： 大脑代谢必须适应广泛的动态范围的能量需求，从时间到时间，从 细胞类型到细胞类型。尽管对大脑活动增加的代谢反应是众所周知的 尽管功能性MRI信号，但这种代谢反应的性质仍然存在很大争议。已经 表明代谢在包括癫痫在内的几种神经系统疾病中起着关键的调节作用 和神经退行性疾病。然而，关于神经元兴奋性耦合的机制知之甚少 代谢，虽然主要的代谢途径已经建立了几十年。 脑代谢的实时动力学的研究受到空间和 时间分辨率的方法，但这样的限制，现在可以克服使用遗传- 编码荧光生物传感器。我将使用传感器的组合，以确定葡萄糖消耗以及 分别以NADH/NAD+和ATP/ADP比率表示。这些传感器可以提供校准的定量读数 在完整的脑组织中，通过使用具有比率或荧光寿命的双光子显微镜， 成像（FLIM）。Ca 2+指标RCaMP 1h和电生理记录将为我提供读数 神经元活动。海马CA 3神经元可以被突触兴奋（这也参与星形胶质细胞）或 逆向地 这项研究结合了荧光葡萄糖、ATP、NADH和Ca 2+生物传感器的使用， 先进的电生理学和光学双光子成像技术，以研究能量代谢， 神经元和星形胶质细胞。目的1是表征葡萄糖、NADH和ATP的动力学 静息时海马切片中星形胶质细胞和神经元（谷氨酸能和GABA能）中的水平 积极的国家。这项工作还将探讨糖酵解对神经元活性诱导的神经元活性的贡献。 加速能量代谢。目的2是确定神经元活动与神经元活动之间的联系机制。 代谢增加，触发刺激（Na+，K+和Ca 2+通量）和随后的信号传导（如 离子型或G蛋白偶联受体和蛋白激酶）。

Project Abstract: Brain metabolism must accommodate a wide dynamic range of energy demands from time to time and from cell type to cell type. Although the metabolic response to increased brain activity is the basis of well-known functional MRI signals, the nature of this metabolic response is still very controversial. It has been demonstrated that metabolism plays a key regulatory role in several neurological conditions, including epilepsy and neurodegenerative diseases. However, little is known about the mechanisms coupling neuronal excitability to metabolism, though the main metabolic pathways have been well established for decades now. The study of real-time dynamics of brain metabolism has been hampered by the insufficient spatial and temporal resolution of the methods, but such limitations can now be overcome by the use of genetically- encoded fluorescent biosensors. I will use a combination of sensors to determine glucose consumption as well as the NADH/NAD+ and ATP/ADP ratios, respectively. These sensors can give calibrated quantitative readouts within intact brain tissue, by using two-photon microscopy with either ratiometric or fluorescence lifetime imaging (FLIM). The Ca2+ indicator RCaMP1h and electrophysiologic recordings will provide me with a readout of neuronal activity. Hippocampal CA3 neurons can be excited synaptically (which also engages astrocytes) or antidromically. The proposed research combines the use of fluorescent glucose, ATP, NADH and Ca2+ biosensors with advanced electrophysiological and optical 2-photon imaging techniques to study the energy metabolism of neurons and astrocytes in brain slices. Aim 1 is to characterize the dynamics of glucose and NADH and ATP levels in the astrocytes and neurons (both glutamatergic and GABAergic) in hippocampal slices during resting and active states. This work will also explore the glycolytic contribution to the neuronal activity-induced acceleration of energy metabolism. Aim 2 is to determine the mechanisms linking the neuronal activity to an increased metabolism, both the triggering stimuli (Na+, K+ and Ca2+ fluxes) and subsequent signaling (such as ionotropic or G-protein coupled receptors and protein kinases).